The synthesis of networks of molecules to perform well-defined functions in cells is a central aim of synthetic biology (Gibbs, W. W., Sci Am 290, 74-81 (2004), Brent, R, Nat Biotechnol 22, 1211-1214 (2004)). Networks have been assembled, or evolved, from a handful of well-characterized natural transcription factors and their binding sites (Basu, S., Gerchman, Y., Collins, C. H., Arnold, F. H. & Weiss, R., Nature 434, 1130-1134 (2005), Elowitz, M. B. & Leibler, S., Nature 403, 335-338 (2000), Gardner, T. S., Cantor, C. R. & Collins, J. J., Nature 403, 339-342 (2000), Guet, C. C., Elowitz, M. B., Hsing, W. & Leibler, S., Science 296, 1466-1470 (2002), Kaern, M., Blake, W. J. & Collins, J. J., Annu Rev Biomed Eng 5, 179-206 (2003), Kobayashi, H. et al., Proc Natl Acad Sci USA 101, 8414-8419 (2004), Yokobayashi, Y., Weiss, R. & Arnold, F. H., Proc Natl Acad Sci USA 99, 16587-16591 (2002), You, L., Cox, R. S., 3rd, Weiss, R. & Arnold, F. H., Nature 428, 868-871 (2004)), to create cellular oscillators, toggle switches and logic functions, and to create novel modes of cell-cell communication and cell pattern formation.
Modified ribosomes with an altered or narrowed scope of mRNA substrates have been examined for possible use in expanding the genetic code and for the purposes of post-transcriptional gene regulation. Previous work has described “specialized ribosomes” (Hui, A. S., Eaton, D. H. & de Boer, H. A., EMBO J. 7, 4383-4388 (1988), Hui, A., Jhurani, P. & de Boer, H. A., Methods Enzymol 153, 432-452 (1987), Hui, A. & de Boer, H. A., Proc Natl Acad Sci USA 84, 4762-4766 (1987)) that bear three mutations in the SD sequence and translate mRNAs bearing complementary mutations in the ASD.
Lee et al. describe experiments in which random mutations were simultaneously introduced to the rRNA binding sequence (SD) on chloramphenicol acetyltransferase mRNA and the complementary message-binding sequence of the E. coli 16S ASD (Lee et al., 1996, RNA 2: 1270-1285). Alternate SD sequences that rely to varying degrees for their translation on wild-type ribosomes were isolated from a collection of ASD and SD mutants (Lee, K., Holland-Staley, C. A. & Cunningham, P. R., RNA 2, 1270-1285 (1996).